A series of proline analogs have been analyzed for their effects on collagen synthesis inhibition in cultures of primary DMBA-induced rat mammary tumors and for their effects on mammary tumor growth in tumor bearing animals. Azetidine carboxylate, thioproline and cis-hydroxyproline were found to be potent, selective inhibitors of collagen synthesis, blocking proline incorporation into collagen by 7 to 27 fold more than incorporation into tumor cell protein. In vivo all 3 of these compounds at doses of 50-200 mg/kg S.C. caused tumor growth arrest or regression. Two possible reasons for these effects on tumor growth seem likely: the tumor cells require collagen synthesis for growth and/or tumors are partial proline auxotrophs. Collagen synthesis requirements were assessed by attempting to rescue the tumor cells from proline analog killing by plating tumor cells on various collagen matrices, with negative results. However proline, usually considered a non-essential amino acid was growth stimulatory for tumor cells. A 30% increase in growth rate was observed at 50 Mug proline/ml growth medium. Even though a reduced sensitivity to proline analogs was not seen on plating the tumor cells on collagen substrata, there was an effect of the proline analogs on basement membrane collagen formation by the tumors in vivo. The analyses of total tumor protein showed that tumors treated for 10 days had only 1/4-1/2 as much 3 as 4-hydroxyproline, consistent with the fact that basement membrane collagen, rich in 3-hydroxyproline, was reduced relative to stromal collagen which has very little 3-hydroxyproline. Since these tumors produce only basement membrane collagen, the proline analog effects were likely to have been due to effects on basement membrane collagen formation by the tumor epithelium.